Generally, a fluid bed reactor may comprise a vertical reactor with an inlet, an outlet, a fluid bed of particles (a solid phase), and a liquid. The liquid is introduced at the inlet and dispersed, optionally through a gas head in case of down-flow reactors, on the bed of solid phase particles, which are suspended and fluidised by the liquid. The liquid is conducted through the bed and a pool of reacted and/or unreacted fluid is let out at the outlet.
Up-flow fluid reactors have liquid inlet at or near the bottom of the reactor and solid phase particles of specific gravity larger than that of the liquid. Down-flow fluid bed reactors have liquid inlet at or near the top of the reactor and solid phase particles of specific gravity less than that of the liquid.
The suspended and fluidised solid phase particles may be impermeable to the fluid or completely permeable to the fluid and substances present in the fluid. The suspended and fluidised solid phase particles may further be reactive or may carry immobilised reactive components selected for solid phase chemical or physical processes with one or more components of the fluid in procedures such as enzymatic reactions; fermentation; ion-exchange and affinity chromatography; filtration; adsorption; chemical catalysis; immunosorption; solid-phase peptide and protein synthesis; and microbiological growth of micro-organisms.
In fluid bed reactors partially solving the problems of packed bed columns, i.e. the problems of suspended matter clogging up the solid-phase bed which increases the back pressures and compresses the bed, disturbing the flow through the bed, the solid phase particles are kept in a free, fluid phase by applying a flow having an opposite direction to the direction of the relative movement of the solid phase particles. Thus, solid phase particles having a density larger than the liquid and moving downwards due to gravity may be kept in a free, fluid phase by an upwards flow of liquid. Also, solid phase particles having a density less than the liquid and thus moving upwards may due to buoyancy be kept in a free, fluid phase by a downwards flow of liquid.
In order to carry out solid phase chemical or physical processes in a fluid bed reactor, an even and smooth distribution of fluid in the fluid bed without back-mixing is often desired. However, fluid bed reactors known in the art do not have efficient means known per se to avoid formation of channels as well as unwanted turbulence and back-mixing in the solid phase particle bed. Especially when the task is to distribute the liquid in large industrial scale reactors (e.g. reactors with cross-sectional areas of more than 0.5–1 m2) it becomes increasingly difficult to obtain a satisfactory distribution of the fluid.
An interesting field of application of fluid bed reactors is EBA.
EBA is an abbreviation for Expanded Bed Adsorption. This is a technology used widely within the biotechnology industries, for example for the production of pharmaceuticals and diagnostic products and in particular for the separation and purification of a broad range of bio-molecules, for example enzymes, proteins, peptides, DNA and plasmids from a vast range of extracts and raw materials, many of which are crude and unclarified. The present invention particularly relates to a new way of introducing the fluid to be processed into an expanded bed adsorption contactor.
The term expanded bed is used to describe a special case of a fluid bed (or fluidised bed) where turbulence or back-mixing of the fluid in the suspended bed of solid phase particles is at a minimum. Another term for this situation is that the fluid passes the expanded bed with “plug flow” and thus resembles the flow pattern in a packed bed wherein turbulence and back-mixing are practically absent.
A traditional purification process for a mixture comprising one or several target molecules could be purification on a packed column (i.e. not EBA), however this requires multiple operational steps such as filtration and centrifugation in order to ensure that impurities in suspension and particulates (for example colloids, whole cells, cell walls, protein aggregates) are removed before the mixture is applied to a suitable packed column. These steps are necessary in order to avoid clogging of the packed column. In the packed column, a given chromatographic medium is present for binding of molecule(s) which are generally the target of the purification, but alternatively can also be used for binding impurities. This chromatographic medium can be adapted to various purification purposes.
The main principle in EBA is to keep the chromatographic medium fluidised and thereby allow particulate impurities, suspended solids and colloidal materials to pass through the column. By using the EBA technology, it is in many instances possible to avoid the above-mentioned operational steps (i.e. those used before packed bed chromatography) before application of the raw material to the column. In this manner, time and expenses for these processes are reduced making EBA a valuable technology, which is economically recommendable for the purification of a large number of different bio-molecules. In addition, productivity and overall yield can be expected to be improved when compared to traditional processing using packed beds.
In order to utilise the EBA technology, an EBA column used to contain a suitable chromatographic medium is required in conjunction with a suitable fluid distribution mechanism.
A brief presentation of the steps generally used in the EBA technology will be given in the following (assuming it is an up-flow process with solid phase particles more dense than the fluid).    1. An adequate quantity of solid phase adsorbent is placed in an EBA column (i.e. the fluid bed rector).    2. Fluid flow through the adsorbent from below is initiated by pumping the liquid to be processed through a fluid distributor. The adsorbent is thereby fluidised (expanded).    3. The adsorbent is rinsed in the column and the conductivity (i.e. salt concentration) and pH are adjusted to what is required to allow binding of the target to the adsorbent.    4. Raw material (i.e. the feedstock) is applied to the expanded bed of adsorbents and the target molecule(s) are bound.    5. The remaining raw material is rinsed out from the column using a wash fluid.    6. The target molecule is eluted off the adsorbent medium by applying a fluid that weakens the interaction with the adsorbent. The elution of the target molecule may be performed after packing the chromatographic adsorbent and reversing the flow direction in the column or the elution may be performed in the expanded bed state.    7. The chromatographic adsorbent is finally (optionally) rinsed and regenerated.
Before the raw material is applied to the column, it should be ensured that expansion of the bed of adsorbent media is stable (i.e. that plug flow like fluid rise is obtained in the column without unwanted turbulence or back mixing of the fluid). The most reliable way of checking seems to be determining the number of theoretical plates by examining the residence time distribution following addition of a tracer. Such methods are well known to those familiar with examining the performance of reactors and particularly to those versed in the art of expanded bed adsorption (for example see the hand book “Expanded Bed Adsorption” by Pharmacia Biotech, Edition AA, page 14; or Levenspiel, O. 1999. Chemical reaction engineering, 3rd ed. John Wiley and Sons, Inc. New York). A satisfying total number of theoretical plates in a column indicating a low degree of back-mixing and fluid flow characteristics suitable for an EBA process is generally in the range of 25 to 30 plates or more (see for example Expanded Bed Adsorption” by Pharmacia Biotech, Edition AA, page 16). In addition, it is generally considered that 50–100 theoretical plates per meter sedimented bed height is satisfactory. Furthermore, visual inspection of the bed particularly using dyes or colored tracers can provide valuable qualitative information about the presence of channels or jet streams in the column. If the solid phase media move only in small circles, channels are not observed and local jet streams of fluid largely devoid of media cannot be seen, this is a good indication that the adsorbent media is expanded in a stable manner.
If an EBA column is not expanded in a stable way and has a plug flow fluid rise, the adsorption efficiency may be low and the whole process economy may be impaired. In analogy, the same will be true for a broad range of other fluid bed and expanded bed reactions not related to adsorption e.g. (continuous) enzymatic reactions and chemical catalysis, chemical synthesis of e.g. peptides and polynucleotides. In these cases, an unstable expansion with turbulence and back-mixing may result in a high loss of unreacted chemical building blocks at the outlet of the column and potentially a slower reaction rate. Also, chemical reaction equilibrium may not be reached or may shifted in an unfavourable direction.
Further information about EBA technology can be found in the book “Expanded Bed Adsorption” by Pharmacia Biotech, Edition AA.
Description of Prior Art
U.S. Pat. No. 4,032,407 discloses a tapered bed bioreactor applying immobilised biological catalysts or enzymatic systems on fluidizable particulate support materials consisting of coal, alumina, sand, and glass, i.e. materials heavier than the fluid, particularly an aqueous fluid.
EP-A-0175568 discloses a three phase fluidised bed bioreactor process comprising purifying effluents in a three phase fluidised bed comprising solid particles being made by mixing a binder with an inorganic material based on aluminum silicate, granulating the resulting mixture, and firing the granules to sinter them. The specific gravity of the sintered granules is adjusted to fall into a specific range from 1.2 to 2.0 by varying the mixing ratio of inorganic powdery materials based on aluminum and binders, said sintered granules having a diameter from 0.1 to 5 mm.
EP-A-0025309 discloses a down-flow fluid bed bioreactor applying biota attached to carrier particles consisting of cork, wood, plastic particles, hollow glass beads or other light weight material and having a specific gravity which is less than that of a liquid sprayed onto the upper part of a fluid bed of suspended carrier particles and conducted downward through the bed.
A disadvantage of distributing an introduced liquid in a fluid bed reactor by simple spraying is the formation of channels in the bed by fluid streams.
EP-A-0005650 discloses an up-flow fluid bed reactor having fluidising liquid flow distributors at the bottom thereof providing flow paths to avoid turbulence effects. Besides requiring complicated flow paths, a great disadvantage of such a distributor is that it may be clogged by particulate matter.
EP-A2-0088404 discloses a fluid bed reactor system for catalytic polymerisation of olefin monomers composed of a cylindrical reaction vessel equipped with distribution plate and agitator disposed in the fluidised bed above the plate and adapted to cause a rotational flow in the fluidised bed, said distribution plate having many passage holes each covered with a cap having an opening the direction of which varies with the distance from the centre of the plate and faces the same direction as the direction of the rotational flow. The fluid bed reactor system is intended to reduce various troubles such as blocking of the distribution plate, formation of polymer agglomerates, and stagnation, adhesion, and agglomeration of polymer at the caps.
EP-A1-0007783 discloses a control system for preventing accumulation of excessive cellular material in a fluidised bed reactor comprising a separator column having means to effect shearing of excess cellular material from the particles to produce in the column a mixture of sheared material and partially stripped carrier particles, said carrier particles being returned to the bed while the sheared material is discharged from the column through the draw-off port. In a specific embodiment the shearing is effected by an agitator arrangement comprising a motor-driven mixing blade operating within the lower portion of the separator column; to rotary speed of the mixing blade being adjusted to an optimum degree of shear for the cellular growth. Excessive pulverisation of the sheared material is avoided by using not a too high rotary speed.
Patent Abstract of Japan, Vol. 8, No. 162, C235 (Abstract of JP 59-62 339) discloses a vertically movable agitator for gas fluidisation equipment to obtain an effective treatment of powder and granules.
EP-A2-0243845 discloses a fluid bed having a built-in device in form of a perforated plate and/or net for performing gas-solid phase reactions whereby generated voids are destroyed so that a homogeneous fluid bed without large voids is provided.
DK/EP 0538350 T3 discloses chromatographic adsorption particles having covalently bound thereto at least one active substance for binding of molecules in a liquid chromatographic fluid bed process. These adsorption particles are formed of a porous composite material with pores permitting access for the said molecules to the interior of the composite material. The spheres can be produced having a given density and diameter. The density is controlled by incorporation of one or more inert particles in the chromatographic medium, the number, material and percentage of the inert particles being significant for the ultimate density of the chromatographic medium. In addition, the pore size can be controlled. The density controlled particles can be viewed as inert heavy/light particles coated with a hydrophilic layer, a conglomeration compound such as an agarose layer of different concentration and thus pore size.
The book “Expanded Bed Adsorption” by Pharmacia Biotech, Sweden, discloses that the size and density of the individual sphere at a given flow situates the sphere at a specific position in the column. The small and light spheres will move to the upper part of the expanded matrix while large, heavy particles will move towards the lower part. The result is that the particles settle at their ideal position after a suitable period of time. When this has taken place, expansion will be stable.
DK/EP 0538350 T3 further discloses a liquid bed reactor as a down/upflowing liquid fluid bed reactor comprising a vertical reactor container with an inlet, an outlet, a fluidised particle bed of chromatographic adsorbent particles and means for initiating movement which are located near by or in the fluidised particle layer which is closest to the liquid inlet. There is a mixed zone, i.e. a stirring zone, the size of which is determined by the degree of stirring, the liquid flow and the quantity of matrix in the reactor container. Above/below this zone is a non-mixed zone in which a so-called plug flow is achieved. By the term plug flow is understood a movement of the liquid as a band through the container and consequently also through the matrix.
An example of such a reactor container is an UpFront column 20™ which is an up-flow reactor developed by UpFront Chromatography A/S, Copenhagen, Denmark.
This reactor container is constructed in such a manner that a supporting net with a pore size of 50 μm is located at the bottom. Below the supporting net is an outlet/inlet which is primarily used as an outlet during elution. A motor axis on which a stirrer is secured extends down the middle of this net. The rate of the stirrer can be varied. Stirring only occurs when the flow comes up through the column. During elution the stirrer is stopped. Right above the supporting net a side inlet is located. Here, all liquid is supplied when the matrix is to be and has been fluidised. This inlet can be opened and closed by sliding the inlet valve into or out of the column pipe. The column pipe is a borosilicate pipe of 20 mm. The actual inflow takes place through four round openings with a diameter of 3 mm each located in that part of the inlet valve which is inside the column pipe. The valve is closed at the end and the four round openings are distributed in the same cross-section in two axes placed at an angle of 90 degrees to each other. The column pipe is 50 cm long (high) and on its side is a scale so as to enable reading of the expansion of the matrix at any time. In addition, the column is provided with a float adapter, an UpFront float, which provides a gentle and good distribution of the elution buffer during (down flow) elution. At the top is an outlet/inlet. Every inlet and outlet is provided with valves on which suitable hoses are mounted. Buffer and raw materials are pumped into the column at an even flow. Typically, the matrix will be ⅓ of the column height. In this case, it is possible to up expand the bed to 3 times. Depending on the type of particles/matrix applied, the flow can vary from 6 column cm/min to 900 column cm/min.
The stirring zone varies from 2–20 cm. In this application the term stirring zone is to be understood as exactly the height in the column at which a stirring of liquid and matrix occur. The viscosity and flow of the liquid and the stirrer's design and rate are significant for the extent of the zone. In addition, it is important that the column is plumb (i.e. vertical). This concept can also be scaled-up to a larger column diameter.
WO 95/20427 discloses a construction for adsorption/desorption of a substance where liquid can flow through a column of matrix. This construction comprises: a) a bottom adapter which is located at the bottom part of the container. The bottom adapter defines the bottom. The adapter has an opening in the bottom for inflow/outflow of liquid to and from the bottom part of the container. This adapter also has a distribution function. It creates the back pressure necessary to create plug flow; b) a top adapter which is located at the top part of the container. This adapter has an opening pointing towards the bottom for inflow/outflow of liquid to and from the top part of the container. It also has a distribution function. This upper adapter has a density permitting that it floats on the liquid passing through the container. By means of hoses, both adapters can lead liquid to and from the container depending on the direction in which the liquid should flow.
Pharmacia Biotech has developed an EBA column which distributes the liquid in another way than by stirring. In the bottom of the column there is an inlet/outlet.
Above the column is a distribution plate through which the liquid has to pass to enter the column. The distribution plate creates the pressure drop necessary to create a plug flow. By the term plug flow is understood the movement of the liquid as one front through the matrix. This bottom adapter leads the liquid vertically upwards through the column. The top adapter can be positioned anywhere necessary in the column. In this manner head space can be reduced. By the term head space is understood the liquid above the matrix.
A serious technical problem in connection with EBA columns using a distribution plate through which the fluid to be processed must pass, is fouling. This is particularly problematic when feedstocks containing for example, whole cells, disrupted cells, particulates, nucleic acids or colloidal materials are present and can lead to blockage of the distribution plate. Another further technical problem is that the distribution plate must have hole sizes small enough to prevent ingress of the solid phase support medium into the fluid distribution mechanism when fluid flow is stopped. This severely limits the lower size of solid phase supports that can be used for fluid treatment.
Although problems of fouling might be addressed by using large (relative to those in a perforated plate distributor) fluid inlet ports in the column base or radially on the column wall and a local mixer to distribute the fluid, a serious technical problem in connection with such known EBA column using a stirrer is that when the column is scaled-up, scale effects begin to play a dominant role. This is most easily recognised by the presence of dead areas/volumes, i.e. areas/volumes where the solid phase medium is not properly contacted with the fluid to be treated. This can lead to channels in the expanded bed and serious reductions in process performance as well as difficulties for cleaning in place and support regeneration. In addition the ability to create a small, localised, well-mixed area in the bottom of a column in the vicinity of the fluid inlet port(s) becomes increasingly difficult as column diameter is increased.